A poly-3-hydroxyalkanoic acid (hereinafter referred to collectively as “PHA”) is a thermoplastic polyester which is synthesized and accumulated as an energy storage substance in cells of a variety of microorganisms and has biodegradability. These days waste plastics are disposed of by incineration or landfill but there are several problems in these disposal methods, such as global warming and ground loosening of reclaimed lands. Therefore, with the growing public awareness of the importance of plastics recycling, ways and means for systematized recycling are being developed. However, uses amenable to such recycling are limited. Actually the disposal load of waste plastics cannot be completely liquidated by said incineration, landfill, and recycling but rather a large proportion of the disposal load is not disposed of but simply left in nature. There is accordingly a mounting interest in PHA and other biodegradable plastics which, after disposal, would be incorporated into the natural cycle of matters and degradation products of which would not exert ecologically harmful influences, and their practical utilizations are highly desired. Particularly a PHA which a microorganism synthesizes and accumulates in their cells is taken up in the carbon cycle of the natural world and it is, therefore, predicted that it will not have any appreciable adverse effects on the ecosystem. Also in the field of medical treatment, it is considered possible to use a PHA as an implant material which does not require recovery or a vehicle for a drug.
Since the PHA synthesized by a microorganism usually forms granules and is accumulated intracellularly, exploitation of the PHA as a plastic requires a procedure for separating it from microbial cells. The known technology for the separation and purification of PHA from microbial cells can be roughly classified into technologies which comprise extracting a PHA from the cells with an organic solvent solving PHA and technologies which comprise removing the cell components other than PHA by cell disruption or solubilization.
Referring to the separation and purification technology of a PHA involving an extraction with an organic solvent, an extraction technique utilizing a halogen-containing hydrocarbon, such as 1,2-dichloroethane or chloroform, as the solvent solving a PHA is known (refer to Japanese Kokai Publication Sho-55-118394 and Japanese Kokai Publication Sho-57-65193). However, since these halogen-containing hydrocarbons are hydrophobic solvents, a pre-extraction procedure, such as drying cells in advance or otherwise, allowing the solvent to directly contact the intracellular PHA is required. Moreover, in such a technology, dissolving PHA at a practically useful concentration (for example, 5%) or higher gives only an extract which is so highly viscous that it involves considerable difficulties in separating the undissolved residues of microbial cells from the PHA-containing solvent layer. Furthermore, in order that PHA may be reprecipitated from the solvent layer at a high recovery, some PHA-insoluble solvent, such as methanol or hexane, need to be used in a large quantity, e.g. 4 to 5 volumes based on the solvent layer, and thus a vessel of large capacity is required for reprecipitation. In addition, the necessary quantity of solvents is so large that both the solvent recovery cost and the cost of lost solvents are enormous. Furthermore, since the use of organohalogen compounds tends to be limited these days for protection of the environment, industrial application of this technology has many obstacles to surmount.
Under the circumstances, there has been proposed an extraction technology using a solvent which is not only capable of dissolving PHA but also miscible with water, for example a hydrophilic solvent such as dioxane (refer to Japanese Kokai Publication Sho-63-198991), propanediol (refer to Japanese Kokai Publication Hei-02-69187), or tetrahydrofuran (refer to Japanese Kokai Publication Hei-07-79788). These methods appear to be favorable partly because PHA can be extracted not only from dry cells but also from wet cells and partly because precipitates of PHA can be obtained by mere cooling of the solvent layer separated from the microbial cell residues. However, even with these methods, the problem of high viscosity of the PHA-containing solvent layer remains to be solved. In addition, while heating is required for enhancing the extraction efficiency, the heating in the presence of water unavoidably results in a decrease in molecular weight and a poor recovery of PHA.
On the other hand, as the technology of removing the cell components other than PHA by solubilization for separation of PHA, J. Gen. Microbiology, vol. 19, 198-209 (1958) describes a technology which comprises treating a suspension of microbial cells with sodium hypochlorite to solubilize cell components other than PHA and recovering PHA. This technology is simple process-wise but the necessity to use a large amount of sodium hypochlorite is a factor leading to a high production cost. Moreover, in view of the marked decrease in molecular weight of PHA and the appreciable amount of chlorine left behind in PHA, this technology is not considered to be suitable for practical use.
Japanese Kokoku Publication Hei-04-61638 describes a process for separating PHA which comprises subjecting a suspension of PHA-containing microbial cells to a heat treatment at a temperature of 100° C. or higher to disrupt the cellular structure and, then, subjecting the disrupted cells to a combination treatment with a protease and either a phospholipase or hydrogen peroxide to solubilize the cell components other than PHA. This technology is disadvantageous in that because the heat treatment induces denaturation and insolubilization of the protein, the load of subsequent protease treatment is increased, that the process involves many steps and is complicated, and that it costs much due to the use of relatively expensive enzymes.
As a technology for disrupting PHA-containing microbial cells, there also has been proposed a method which comprises treating microbial cells with a surfactant, decomposing the nucleic acids released from the cells with hydrogen peroxide, and separating PHA (refer to Japanese Kohyo Publication Hei-08-502415) but the waste liquor containing the surfactant develops a copious foam and, in addition, has a high BOD load value. From these points of view, the use of a surfactant is objectionable for production on a commercial scale.
There has also been proposed a technology for separating PHA which comprises disrupting PHA-containing microbial cells with a high-pressure homogenizer (refer to Japanese Kokai Publication Hei-07-177894 and Japanese Kokai Publication Hei-07-31488). However, this technology has the drawback that PHA with high purity cannot be obtained unless a suspension of microbial cells is subjected to a high-pressure treatment at least 3 times, or 10 times, if necessary, with heating, and even then the purity of thus-obtained PHA that can be attained is as low as about 65 to 89%.
There has also been proposed a technology for separating PHA which comprises adding an alkali to a suspension of PHA-containing microbial cells, heating the suspension, and disrupting the cells (refer to Japanese Kokai Publication Hei-07-31487). However, this technology is disadvantageous in that the purity of the product polymer that can be attained is as low as 75.1 to 80.5% and that if the level of addition of the alkali be raised to improve the yield, the molecular weight of the polymer would be decreased. Several techniques for carrying out physical disruption after addition of an alkali have been proposed (refer to Japanese Kokai Publication Hei-07-31489 and Bioseparation, 1991, vol. 2, 95-105) but since the alkali treatment alone results in the extracellular release of only a small amount of cell components and some of such cell components are retained in the PHA fraction even after subsequent high-pressure disruption treatment, these techniques are invariably inefficient. Thus, PHA of high purity cannot be separated unless the microbial cell suspension is subjected to at least 5 cycles of high-pressure treatment and even then the purity of PHA is as low as about 77 to 85%. The technology involving addition of an alkali has an additional drawback; generally the cell components released from microbial cells, particularly nucleic acids, increase the viscosity of the cell suspension to make subsequent processing difficult.
There has also been proposed a technology in which a suspension of PHA-containing microbial cells is adjusted to an acidity lower than pH 2 and PHA is separated at a temperature not below 50° C. (refer to Japanese Kokai Publication Hei-11-266891). However, this technology is disadvantageous in that the treatment under the strongly acidic condition below pH 2 is undesirable for production on a commercial scale, that the acid treatment needs to be followed by adjustment to the alkaline side for enhanced purity but this entails massive salt formation, and that the molecular weight of the product PHA is decreased from 2,470,000 to about 1,000,000.
Japanese Kokai Publication 07-177894 proposes a technology for separating and purifying poly-3-hydroxybutyrate (hereinafter referred to as PHB) by treating microbial cells with an oxygen bleach after conducting a high-pressure disruption treatment. Although a method for treating PHB slurry with various oxygen bleaches is disclosed, there is no description about pH in the bleaching treatment.